PROTEINS MODIFICATIONS FOSTERED BY THE PRO-OXIDANT ENVIRONMENT OF PARKINSON¿S AND ALZHEIMER¿S DISEASES CEREBROSPINAL FLUID: THE CERULOPLASMIN CASE

In the last decades the role of metals in neurodegenerative diseases assumed a great importance, in particular regarding copper and iron. Ceruloplasmin (Cp), an enzyme present in the plasma secreted by the liver and in the cerebrospinal fluid (CSF) as product of the epithelial cells composing the blood-CSF barrier, may play a crucial function in the metabolism of copper and iron in the central nervous system (CNS), being Cp able to transport copper and to oxidize toxic ferrous iron; Cp’s ferroxidasic function is essential for iron export outside cells of the CNS, thus protecting tissues from oxidative damage. Investigating Cp patterns using two-dimensional electrophoresis (2D-E), we found that in Parkinson’s (PD) and Alzheimer’s (AD) diseases the Cp profile proved to be more acidic than that found in healthy controls and patients affected by other neurological pathologies, suggesting a possible use of Cp 2D-E profile as a pathological marker. In vitro oxidation of Cp generated a 2D-E shift resembling that observed in patients, indicating that Cp oxidation contributes to the electrophoretic profile seen in PD and AD; Cp oxidation caused a decrease of ferroxidase activity, promoting intracellular iron retention in cultured neurons. Among all the possible Cp’s amino acidic modifications induced or accelerated by oxidation, the deamidation of asparagine appears attractive, being this reaction related to protein aging, dis-function, degradation and/or aggregation, all phenomena observed for several proteins in AD and PD. In particular our attention has been focused on the asparagine present at the level of the two Asp-Gly-Arg (NGR) motifs present on the Cp sequence; the first, 568NGR, is exposed on the surface and in direct contact with the environment while the second 962NGR, is hidden within the tertiary structure of the protein. The function of NGR-motif has been recently discovered: deamidation of the asparagine at this level results in the formation of isoDGR-motif that can bind integrins containing RGD-binding site. This open a new interesting research field on latent integrin-recognition-sites, which can be activated in physiological or pathological conditions. We observed that the Cp exposed 568NGR site can deamidate under conditions mimicking accelerated asparagine aging, while the hidden 962NGR-site can deamidate exclusively when aging occurs under oxidative conditions; this result suggest that oxidation-induced structural changes foster deamidation at this site. NGR-deamidation in Cp was associated with gain of integrin binding function, intracellular signalling and cell pro-adhesive activity. Moreover, deamidated-Cp intracellular signals in epithelial and neural stem cells caused cell proliferation arrest. In addition, also the ex-vivo aging in the CSF from Alzheimer and Parkinson disease patients, but not in control or peripheral neuropathies CSF, induced Cp structural changes, deamidation with gain of integrin-binding function, carbonylation and ferroxidase-activity reduction. These results indicate that in the pathological CSF are present compounds (small molecules or proteins) able to induce oxidative modifications on other proteins. In particular, the modifications observed in Cp aged in pathological CSF may be the consequence of oxidation resulting from the high H2O2 concentration we detected in the AD’s and PD’s CSF. Concluding, the CSF of AD and PD patients favors Cp deamidation at NGR-sites with gain-of-function in cell adhesion and loss of ferroxidase activity, raising Cp deamidation as an additional contributor to neurodegenerative diseases characterized by oxidation.